1. Field of the Invention
The present invention relates to a method for producing polyhydroxyalkanoate (which is also referred to as xe2x80x9cpoly-3-hydroxyalkanoic acidxe2x80x9d, and hereinafter abbreviated as xe2x80x9cPHAxe2x80x9d at times), using a microorganism having an ability to produce and accumulate PHA in a cell thereof, or a higher organism such as a plant cell, in which PHA production has enabled by introduction of a PHA synthesis gene.
More specifically, the present invention relates to a method for producing PHA, which comprises a step of removing cell components other than PHA by treating cells containing PHA with an oxidizing agent, which can efficiently remove cell components other than PHA with a few steps, at a low cost, and obtain highly purified PHA with high yield. Further, the present invention relates to a method for producing PHA, wherein PHA containing a reduced amount of residual chlorine or no residual chlorine can be obtained.
Furthermore, the present invention relates to an apparatus, with which the above-described methods for producing PHA can be carried out.
2. Related Background Art
Up till now, it has been reported that a large number of microorganisms produce and accumulate poly-3-hydroxybutyric acid (hereinafter abbreviated as xe2x80x9cPHBxe2x80x9d at times) or other PHAs in cells thereof (Biodegradable Plastic Handbook, ed. by A Study Group of Biodegradable Plastics, NTS, Inc., p. 178-197). As the conventional plastics, these polymers can also be used for production of various products by melt processing, etc. Moreover, these polymers have an advantage of biodegradability to be completely decomposed by microorganisms in the nature. Therefore, unlike many synthetic high polymers previously used, these polymers do not remain in the natural environment and cause no pollution, and they require no incineration treatment, so they can be useful materials in terms of prevention of air pollution or global warming. Further, these polymers are excellent in biocompatibility, and so it is expected that they will be applied as medical soft materials, etc. It is known that the PHA produced by microorganisms can have various compositions or structures depending on the type of microorganisms used for the production, the composition of medium, culture conditions, etc., and researches on the control of the composition or structure have been made mainly from the viewpoint of improvement of the physical properties of PHA.
For example, it has been reported that Alcaligenes eutrophus H16 (ATCC No. 17699) and mutants thereof produce a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid in various composition ratios by varying carbon sources when they are cultured (Japanese Patent Publication Nos. 6-15604, 7-14352, 8-19227, etc.). Japanese Patent Application Laid-Open No. 5-74492 discloses a method for allowing microorganisms of Methylobacterium sp., Paracoccus sp., Alcaligenes sp., Pseudomonas sp. to produce a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid by contacting them with primary alcohol containing 3 to 7 carbon atoms. Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonas acidovorans IF013852 produces a polyester having 3-hydroxybutyric acid unit and 4-hydroxybutyric acid unit by performing culture using gluconic acid and 1,4-butanediol as carbon sources.
Japanese Patent No. 2642937 discloses that Pseudomonas oleovorans ATCC 29347 produces PHA having a 3-hydroxyalkanoic acid unit containing 6 to 12 carbon atoms, where noncyclic aliphatic hydrocarbon is provided thereto as a carbon source. Japanese Patent Application Laid-Open Nos. 5-93049 and 7-265065 disclose that Aeromonas caviae produces a copolymer of two components, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, by performing culture using oleic acid or olive oil as a carbon source.
The above described PHAs are all PHAs consisting of monomer units having an alkyl group on a side chain, which are synthesized by xcex2-oxidation of carbohydrate etc. or fatty acid synthesis from a sugar by a microorganism and each of these PHAs is what is called xe2x80x9cusual PHAxe2x80x9d.
It has been reported that some types of microorganisms produce, PHA having various substituents other than alkyl groups introduced into side chains thereof, that is what is called xe2x80x9cunusual PHAxe2x80x9d, and using such means, an attempt to improve the physical properties of the PHA produced by microorganisms has been started. Moreover, where a broader application of the PHA produced by microorganism, e.g. application as a functional polymer is considered, the xe2x80x9cunusual PHAxe2x80x9d is extremely useful. Examples of substituents include a substituent comprising an aromatic ring (a phenyl group, a phenoxy group, a benzoyl group, etc.), an unsaturated hydrocarbon, an ester group, an allyl group, a cyano group, a halogenated hydrocarbon, an epoxide, etc. Of these, particularly, intensive studies on PHA having an aromatic ring have been progressing.
(a) Substituents comprising a phenyl group or a partially substituted phenyl group
Makromol. Chem., 191, 1957-1965 (1990) and Macromolecules, 24, 5256-5260 (1991) report that Pseudomonas oleovorans produces PHA comprising 3-hydroxy-5-phenylvaleric acid as a unit from 5-phenylvaleric acid as a substrate. Macromolecules, 29, 1762-1766 (1996) reports that Pseudomonas oleovorans produces PHA comprising 3-hydroxy-5-(4xe2x80x2-tolyl)valeric acid as a unit from 5-(4xe2x80x2-tolyl)valeric acid as a substrate. Macromolecules, 32, 2889-2895 (1999) reports that Pseudomonas oleovorans produces PHA comprising 3-hydroxy-5-(2xe2x80x2,4xe2x80x2-dinitrophenyl)valeric acid and 3-hydroxy-5-(4xe2x80x2-nitrophenyl)valeric acid as units from 5-(2xe2x80x2,4xe2x80x2-dinitrophenyl)valeric acid as a substrate.
(b) Substituents comprising a phenoxy group or a partially substituted phenoxy group
Macromol. Chem. Phys., 195, 1665-1672 (1994) reports that Pseudomonas oleovorans produces a PHA copolymer of a 3-hydroxy-5-phenoxyvaleric acid unit and a 3-hydroxy-9-phenoxynonanoic acid unit, from 11-phenoxyundecanoic acid as a substrate. Japanese Patent No. 2989175 discloses inventions regarding: a homopolymer consisting of a 3-hydroxy-5-(monofluorophenoxy)pentanoate (3H5(MFP)P) unit or a 3-hydroxy-5-(difluorophenoxy)pentanoate (3H5(DFP)P) unit; a copolymer comprising at least a 3H5(MFP)P unit or a 3H5(DFP)P unit; and a method for producing the above-described polymers, using Pseudomonas putida of Pseudomonas sp. that synthesizes these polymers. The publication states that the effect of the inventions is to provide stereoregularity and water repellency while retaining a high melting point and good workability. Moreover, Japanese Patent Application Laid-Open No. 2000-72865 reports that Pseudomonas putida 27N01 produces PHA comprising various types of 3-hydroxyfluorophenoxyvaleric acid units.
In addition to such fluorine-substituted forms, studies on cyano- or nitro-substituted forms have also been progressed. Can. J. Microbiol., 41, 32-43 (1995) and Polymer International, 39, 205-213 (1996) report the production of PHA comprising 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit from octanoic acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as substrates, using Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442.
These reports are useful to obtain polymers having physical properties derived from PHA having an aromatic ring on a side chain thereof, unlike a usual PHA having an alkyl group on a side chain.
As a new category, not only regarding change of physical properties, but also a study for producing PHA having a suitable functional group on a side chain thereof to create a new function using the functional group, is progressing.
For example, Macromolecules, 31, 1480-1486 (1996), Journal of Polymer Science: Part A: Polymer Chemistry, 36, 2381-2387 (1998), etc. report that PHA comprising a highly reactive epoxy group at the terminus of a side chain thereof was synthesized by synthesizing PHA comprising a unit having a vinyl group at the terminus of the side chain and then performing epoxidation with an oxidizing agent. Further, other than a vinyl group, as an example of synthesizing PHA comprising a unit having thioether that is expected to have a high reactivity, Macromolecules, 32, 8315-8318 (1999) reports that Pseudomonas putida 27N01 produces a PHA copolymer comprising a 3-hydroxy-5-thiophenoxyvaleric acid unit and a 3-hydroxy-7-thiophenoxyheptanoic acid unit from 11-thiophenoxyvaleric acid as a substrate.
It is known that microorganisms accumulate the produced PHA in their cells in the form of particles. To separate the PHA accumulated in the cells from microorganism cells and purify it, there are both a method involving extraction of PHA with a chlorine containing organic solvent such as chloroform or dichloromethane and a method for obtaining PHA particles by solubilizing cell components other than PHA for removal. The former solvent extraction method is an excellent method for simply extracting and separating PHA with high purity, but at an industrial production level, production scale increases, causing a problem regarding a need for a large amount of organic solvent (it is pointed out that a chlorine containing organic solvent used such as chloroform causes an environmental or health problem when the solvent volatilizes or transpires). Moreover, when PHA should be obtained in the form of particles, the method involving solvent extraction can not be employed.
On the other hand, as the latter method involving solubilization of cell components other than PHA for removal, there is known a method comprising solubilizing cell components other than PHA by a treatment with an agent such as hypochlorite or hydrogen peroxide, and collecting insoluble PHA particles by solid-liquid separation. For example, J. Gen. Microbiology, 19, 198-209 (1958) reports a method for separating and purifying polymers by treating microorganism cells with an alkali solution of sodium hypochlorite. Japanese Patent Application Domestic Publication No. 8-508881 discloses a method for separating PHA from a cell by treating a PHA accumulating cell with endopeptidase, then treating with a suitable chelating agent, and further treating with hydrogen peroxide.
As another method, Japanese Patent Application Laid-Open No. 57-174094 discloses a method for separating PHA from a microorganism cell by heating and pressurizing a PHA accumulating cell, and then releasing the pressure to crush the cell. Japanese Patent Application Laid-Open No. 63-226291 discloses a method for separating PHA, which comprises converting cell bodies to spheroplasts, crushing them by sonication, performing centrifugal separation, and separating PHA in the uppermost layer formed after the centrifugal separation.
These methods are extremely useful even when PHA is required to be obtained in the form of particles.
However, in the above-stated treatments, there are problems that operation is complicated and that enzyme, acid or alkali is used and is likely to remain after the use therefore a method for more simply collecting PHA with high purity is desired.
When compared with methods using other treating agents, a method using an oxidizing agent such as hypochlorite or hydrogen peroxide, needs moderate treatment conditions with simple steps, and impurities from cells are seldom mixed, so highly purified PHA can be obtained in the form of particles. Thus, this is an excellent method having many practical advantages such as low cost, but it has problems to be solved as stated below.
For example, it has been considered that a method for collecting PHA accumulated in cells in the form of particles by treating the cells with hypochlorite such as sodium hypochlorite, is not adequate to practical use, since it has problems that molecular weight is reduced depending on the structure of PHA, and that a considerable amount of chlorine is remained in the collected PHA (for example, see Japanese Patent Application Laid-Open No. 7-177894).
According to studies of the present inventors, it was found that this residual chlorine is released quickly, when PHA is heated. So, when a desired processed product is made from PHA particles obtained by using such a hypochlorite treatment, if a large amount of chlorine is released in a heating process thereof, it is strongly concerned that the released chlorine becomes a factor of pollution of work environment, etc. In addition, according to studies of the present inventors, it has also been clarified that the residual chlorine contained in the PHA particles cannot be removed sufficiently by a usual treatment such as washing with water. A conclusion is reached from the studies that, although the residual chlorine is strongly retained by PHA particles, it is released at a time by performing a heating process, and thereby the above-stated large amount of chlorine is observed.
Accordingly, in a method for producing PHA involving a treatment with hypochlorite in the production process, the development of means for reducing residual chlorine strongly retained by PHA particles is an extremely important challenge in developing and using PHA on a commercial scale, especially in obtaining and using the PHA in the form of particles.
On the other hand, in a method for collecting PHA accumulated in cells by treating the cells with a peroxide compound such as hydrogen peroxide or sodium peroxide, increase of purity of the collected PHA is an extremely important challenge in developing and using PHA on a commercial scale.
Therefore, an object of the present invention is, when PHA is produced by treating with an oxidizing agent, to solve the above-stated problems in the prior art and to provide a method for producing PHA which comprises efficiently removing cell components other than PHA with a few steps, at a low cost, so as to obtain highly purified PHA with high yield.
Moreover, another object of the present invention is, when PHA is produced by treating with an oxidizing agent containing hypochlorite, to provide a method for producing PHA which comprises a step of removing or reducing chlorine remained in the collected PHA particles by simple means with a good reproducibility.
As a result of thorough studies on a method for producing highly purified PHA with high yield by efficiently removing cell components other than PHA from cells containing PHA, with a few steps, at a low cost, the present inventors have accomplished the following invention.
That is to say, the present invention relates to a method for producing PHA by treating cells containing PHA with an oxidizing agent to remove cell components other than PHA, which comprises a step of treating the cells with an oxidizing agent comprising at least hypochlorite, a step of separating the treated cells into a water-soluble fraction and a water-insoluble fraction, and a step of reducing chlorine remained in the water-insoluble fraction.
Specifically, the present invention relates to a method for producing PHA by treating cells containing PHA with an oxidizing agent to remove cell components other than PHA, which comprises a step of treating the cells with an oxidizing agent comprising at least hypochlorite, a step of separating the treated cells into a water-soluble fraction and a water-insoluble fraction, and a step of washing the water-insoluble fraction with a hot water.
Moreover, the present invention relates to a method for producing PHA by treating cells containing PHA with an oxidizing agent to remove cell components other than PHA, which comprises a step of treating the cells with an oxidizing agent comprising at least hypochlorite and a step of washing the obtained PHA with an aqueous thiosulfate solution.
Furthermore, the present invention relates to a method for producing PHA by treating cells containing PHA with an oxidizing agent to remove cell components other than PHA, which comprises a step of treating the cells with an oxidizing agent comprising at least hypochlorite and a step of washing the obtained PHA with a polar solvent solution comprising at least an organic polar solvent in which the PHA is insoluble.
Still more, the present invention relates to a method for producing PHA, comprising a step of obtaining a crushed product by crushing cells containing PHA, a step of separating the crushed product into a water-soluble fraction and a water-insoluble fraction and a step of treating the water-insoluble fraction with an oxidizing agent.
Still further, the present invention relates to an apparatus with which the above-described method for producing PHA can be carried out.